NIR light-activated nanocomposites combat biofilm formation and enhance antibacterial efficacy for improved wound healing

The study addresses the urgent challenge of rising antibiotic resistance that complicates treatment of severe infections and is exacerbated by antibiotic overuse. Photothermal therapy (PTT) and photodynamic therapy (PDT) have emerged as synergistic strategies to combat multidrug-resistant bacteria, with metal-based nanoparticles (e.g., Cu, Ag, Au) serving as promising agents due to their unique optical properties that facilitate energy and electron transfer to enhance PDT/PTT efficiency. Copper exhibits intrinsic antibacterial activity and can catalyze ROS generation via Fenton-like reactions, but conventional CuO nanoparticles often require high concentrations for bactericidal effects. To overcome these limitations, the authors aim to develop selenium–tellurium doped copper oxide nanoparticles (SeTe-CuO NPs) with dual photothermal and photodynamic capabilities to enhance antibacterial performance, inhibit biofilms, and promote wound healing under NIR irradiation.

Background literature highlights the global threat of antimicrobial resistance and the promise of nanomaterials in antibacterial therapy and wound management. Prior works demonstrate that: (1) combining PTT and PDT enhances antibacterial efficacy against resistant strains; (2) metal-based nanoparticles improve photosensitizer performance via optical and electron transfer effects; (3) copper-based materials possess strong antimicrobial properties, can catalyze ROS from H2O2 via Fenton-like activity, and have been used in coatings and wound dressings with acceptable in vivo biocompatibility; (4) CuO nanoparticles are effective but often need high doses, prompting surface modification strategies to boost activity; and (5) chalcogenide nanostructures (Se, Te, Se-Te) have shown bioactivity and can be integrated into composite systems for synergistic antibacterial effects. This context supports the design of SeTe-CuO nanocomposites to leverage dual PDT/PTT mechanisms for improved antibacterial and wound-healing outcomes.

Synthesis and characterization: Se-Te nanoparticles were synthesized in CTAB-containing aqueous solution using telluric acid and sodium selenite (20 mM each) reduced by hydrazine and ascorbic acid at 95 °C. The product was purified and dried. CuO was then immobilized onto Se-Te NPs by mixing Se-Te NPs (0.3 g in 30 mL water) with 2 mM CuO NP solution (10 mL), stirring at 75 °C for 2 h, followed by hydrazine reduction, purification, and drying to yield SeTe-CuO NPs. Structural and compositional characterization included TEM, EDS, XRD, XPS (Cu 2p, Se 3d, Te 3d), FTIR, and Raman spectroscopy, confirming successful CuO immobilization while maintaining Se-Te crystallinity. Photodynamic and photothermal assessment: ROS generation under 808 nm NIR irradiation (0.8 W cm−2) was measured by EPR using DMPO for •OH and TEMP for 1O2 (detecting TEMPO adduct). Photothermal performance was assessed by irradiating NP suspensions with an 808 nm laser and recording temperature rise via IR thermal camera; photostability was examined through repeated on/off irradiation cycles and calculating photothermal conversion efficiency per a published method. In vitro antibacterial assays: Antibacterial activity against E. coli (ATCC8739) and S. aureus (ATCC6538) was evaluated by agar well diffusion (zones of inhibition), agar plate colony assays with/without NIR irradiation, and bacterial growth curves (OD600) under varying NP concentrations (12–96 µg mL−1). Antibiofilm activity was quantified using crystal violet staining after forming biofilms in 96-well plates and treating with SeTe-CuO NPs (20–100 µg mL−1). Bacterial membrane damage and viability: SEM assessed morphology after NP treatment and NIR exposure. Live/dead staining used DAPI/PI and CLSM imaging after treating bacteria with 48 µg mL−1 SeTe-CuO NPs and NIR irradiation (0.8 W cm−2, 10 min). Intracellular ROS in bacteria was quantified using DCFH-DA (10 µM) with fluorescence measurement (Ex 485 nm/Em 520 nm) for groups: PBS, SeTe-CuO NPs, SeTe-CuO NPs + L. Biocompatibility: Cytotoxicity was tested in L929 mouse fibroblasts (10–100 µg mL−1) by CCK-8 after 24 h. Hemolysis was evaluated by incubating mouse RBCs with SeTe-CuO NPs (25–100 µg mL−1) for 3 h at 37 °C and measuring absorbance at 540 nm to calculate hemolysis percentage. In vivo wound infection model: Female BALB/c mice (5–6 weeks) received 6 mm dorsal full-thickness wounds, inoculated with bacterial suspension (OD600 0.4). After 24 h, treatments were applied every second day: PBS, Se-Te NPs, SeTe-CuO NPs, or SeTe-CuO NPs + NIR (808 nm). Wounds were photographed on days 1, 3, 6, 9, and 12; wound area was quantified with ImageJ to calculate closure percentage. Histology included H&E staining to assess re-epithelialization, collagen, and granulation tissue; Giemsa staining assessed bacterial burden on days 6 and 12; bacteria recovered from wound tissues were cultured on agar to quantify viability. Animal procedures followed approved guidelines (ZYZY2022090055).

• SeTe-CuO NPs exhibited strong ROS generation upon 808 nm irradiation, with pronounced EPR signals for singlet oxygen and hydroxyl radicals, unlike Se-Te NPs.
• Photothermal performance: Under 808 nm irradiation, SeTe-CuO NPs reached 60.2 °C, versus 48 °C for Se-Te NPs; demonstrated excellent photostability across irradiation cycles.
• Antibacterial activity (agar diffusion): Zones of inhibition for SeTe-CuO NPs were 35 ± 2 mm against E. coli and 30.66 ± 2 mm against S. aureus, outperforming Se-Te NPs and CuO NPs.
• Biofilm inhibition: Dose-dependent inhibition with ~80% reduction in E. coli biofilm and ~70% in S. aureus at 100 µg mL−1.
• Growth inhibition: Bacterial growth was suppressed across increasing NP concentrations; plate assays confirmed high antibacterial activity both with and without NIR, enhanced with NIR.
• Membrane disruption: SEM showed significant morphological damage in bacteria treated with SeTe-CuO NPs; CLSM live/dead staining indicated increased red fluorescence (dead cells), especially with NIR.
• Intracellular ROS: Fluorescence intensity increased in the order PBS < SeTe-CuO NPs < SeTe-CuO NPs + L, confirming enhanced ROS generation under NIR.
• In vivo wound healing: In infected mouse wounds, SeTe-CuO NPs + NIR achieved ~93% wound closure by day 12, superior to other groups; reduced scars and enhanced re-epithelialization, granulation tissue, and hair follicle development.
• Bacterial clearance in vivo: Giemsa staining showed markedly fewer bacteria in SeTe-CuO NPs + NIR on day 6 and none detectable by day 12; agar culture from wound tissues corroborated reduced bacterial viability.
• Biocompatibility: L929 cell viability remained >95% at up to 100 µg mL−1; hemolysis ~5% at 100 µg mL−1, indicating good cytocompatibility and blood compatibility.
• Overall, dual PDT/PTT synergy enabled up to ~99% bacterial eradication and significant biofilm suppression under NIR, translating to accelerated wound healing.

The findings demonstrate that integrating CuO with Se-Te to form SeTe-CuO nanocomposites leverages synergistic photothermal and photodynamic effects under 808 nm NIR irradiation to overcome limitations of conventional CuO antibacterial agents. Enhanced ROS generation (singlet oxygen and hydroxyl radicals) and efficient photothermal heating contribute to membrane disruption, intracellular oxidative stress, and bacterial death, which collectively inhibit planktonic growth and biofilm formation. In vivo, the synergy translated into rapid bacterial clearance and significantly accelerated wound closure, supported by histological evidence of improved re-epithelialization, collagen deposition, granulation tissue, and hair follicle formation. The nanocomposites also showed favorable biocompatibility in vitro and in vivo, underscoring their potential as a therapeutic platform for managing infected wounds and combating multidrug-resistant pathogens while potentially lowering dependence on high-dose antibiotics.

A facile one-pot synthesis yielded SeTe-CuO nanoparticles with dual photothermal and photodynamic functionalities. These nanocomposites generated robust ROS and photothermal responses under NIR, producing strong antibacterial and antibiofilm effects against both gram-negative (E. coli) and gram-positive (S. aureus) bacteria. In vivo, NIR-activated SeTe-CuO NPs achieved rapid bacterial clearance and significantly accelerated wound healing with good biocompatibility. Mechanistically, the nanocomposites disrupt bacterial membranes and elevate intracellular ROS, enhancing bactericidal efficacy. The dual/multimodal synergy indicates potential to reduce conventional antibiotic dosages and offers a versatile platform for treating drug-resistant bacterial infections and improving wound management. Future work could explore broader pathogen panels, dosing regimens, long-term biosafety, and integration into wound dressings or injectable formulations.